Phenological segregation suggests speciation by time in the planktonic diatom Pseudo‐nitzschia allochrona sp. nov.

Abstract The processes leading to the emergence of new species are poorly understood in marine plankton, where weak physical barriers and homogeneous environmental conditions limit spatial and ecological segregation. Here, we combine molecular and ecological information from a long‐term time series and propose Pseudo‐nitzschia allochrona, a new cryptic planktonic diatom, as a possible case of speciation by temporal segregation. The new species differs in several genetic markers (18S, 28S and ITS rDNA fragments and rbcL) from its closest relatives, which are morphologically very similar or identical, and is reproductively isolated from its sibling species P. arenysensis. Data from a long‐term plankton time series show P. allochrona invariably occurring in summer–autumn in the Gulf of Naples, where its closely related species P. arenysensis, P. delicatissima, and P. dolorosa are instead found in winter–spring. Temperature and nutrients are the main factors associated with the occurrence of P. allochrona, which could have evolved in sympatry by switching its phenology and occupying a new ecological niche. This case of possible speciation by time shows the relevance of combining ecological time series with molecular information to shed light on the eco‐evolutionary dynamics of marine microorganisms.

with morphological stasis in many instances and leading to an escalation in the discovery of cryptic or pseudocryptic species. The evidence of so far hidden diversity in marine microbes has increasingly emerged with the discovery of multiple species within iconic taxa long considered to be a single one. Notable examples are the diatoms Skeletonema  and Leptocylindrus (Nanjappa et al., 2013) and the prasinophyte Micromonas (Simon et al., 2017).
More evidences have been provided by massive sequencing of environmental DNA, which has revealed high level of interspecific and intraspecific genetic diversity in the microbial realm (de Vargas et al., 2015;Gaonkar et al., 2020;Moon-van der Staay et al., 2001).
Distinct temporal or biogeographic patterns among pseudocryptic and cryptic species indicate that, in spite of morphological stasis, their phylogenetic diversity is also reflected in functional aspects such as their ecophysiological characteristics (Casteleyn et al., 2010;Foulon et al., 2008). At the same time, the discovery of the coexistence of many hardly distinguishable organisms in an apparently homogeneous environment exacerbates the so-called "paradox of plankton" (Hutchinson, 1961), based on the idea that competitive exclusion in such a resource-limited environment as the ocean should favor few fittest species occupying large, unstructured niches. At the global scale, genetic differences within taxa previously considered ubiquitous challenge the view of the prevalence of cosmopolitan microbes that would occur wherever the environment permits-"everything is everywhere, but, the environment selects" (Baas Becking, 1934;de Wit & Bouvier, 2006)-with limited biogeographic patterns and a consequent low diversity (Fenchel, 2005;Fenchel & Finlay, 2004).
The question left open by the findings of the last decades is how that great microbial diversity may arise at sea, and particularly in the plankton, where physical barriers are virtually absent and species' dispersal potential is unlimited. In these conditions, the role of geographic separation in promoting species diversification in allopatry appears unlikely (Palumbi, 1994). Sympatric speciation driven by ecological segregation (Potkamp & Fransen, 2019;Whittaker & Rynearson, 2017) could also be limited for planktonic microalgae in the often remixed photic zone. In the terrestrial habitat, the divergence of breeding times, that is, allochronic segregation, has been posited as a plausible mechanism for sympatric speciation, whereby phenological changes in part of the population can promote assortative mating and genetic divergence between subpopulations (Hendry & Day, 2005;Weis & Kossler, 2004). However, the possibility of sympatric speciation by allochronic segregation has rarely been considered for aquatic organisms (Rosser, 2016), although the existence of a temperature barrier for sexual reproduction between closely related species has been hypothesized to explain sexualization failure in some cases (Amato et al., 2007;Quijano-Scheggia et al., 2009).
The pennate diatoms Pseudo-nitzschia are needle-like, chainforming planktonic organisms thriving in coastal waters around the world's seas. Their life cycle includes heterothallic sexual reproduction, through which these species re-establish maximal cell size (Montresor et al., 2016). The diversity of the genus, now including 58 species (Guiry & Guiry, 2022), has expanded over the years due to the raised attention to the production in some species of neurotoxins (domoic acid, DA) that cause a syndrome known as amnesic shellfish poisoning. The use of molecular markers has much contributed to the description of new species within complexes of taxa that are hardly distinguishable morphologically, not even with electron microscopy (see Lim et al., 2018 for an updated review of the genus). These cryptic and pseudocryptic species may show distinct geographic ranges and temporal patterns Ruggiero et al., 2015), as well as different biochemical and functional traits (Lamari et al., 2013), while intricate phylogenetic relationships and intraspecific genetic variations over the years indicate complex microevolutionary dynamics (D'Alelio & Ruggiero, 2015).
In the Gulf of Naples (GoN), 12 Pseudo-nitzschia species are recorded Zingone et al., 2006). Among them, the P. delicatissima-complex (Lundholm et al., 2006) is the most represented group, with three different species only identifiable with certainty by means of molecular methods: P. delicatissima sensu stricto, P. arenysensis and P. dolorosa. Here, we describe another cryptic P. delicatissima-like species as P. allochrona sp. nov. based on sequences of diagnostic nuclear (18S rDNA, 28S rDNA, and ITS rDNA) and chloroplast (partial RUBISCO, rbcL) markers, ITS2 secondary structure and interbreeding experiments with its sibling species P. arenysensis.
By coupling molecular information with environmental data from a 30 ys-long time series, we build on the phenological and ecological peculiarities of P. allochrona to discuss possible mechanisms of speciation in the plankton realm.  Table S1). Two additional strains were isolated from the Ionian Sea (Mediterranean Sea) in September 2008. In addition, in this study, we considered 187 strains of other P. delicatissima-like species obtained from the Gulf of Naples, which had been identified as P. arenysensis, P. delicatissima or P. dolorosa with molecular analyses in previous studies (Amato et al., 2007;Barra et al., 2013;Orsini et al., 2004), for which we could track the isolation date. Two further strains of P. arenysensis from the Gulf of Naples (BB16 and CM63, courtesy of M. Ferrante, SZN) were used in mating experiments. All strains were isolated by hand pipetting. Cultures were grown in F/2 medium and maintained at 20°C under an irradiance of 70-80 μmol photon m −2 s −1 and a 12:12 light:dark regime.

| Microscopy
Live culture material of P. allochrona was observed and cell measurements taken under a Zeiss Axiophot and an Axiovert 200 light microscopes (Carl Zeiss). Pictures were taken with a Zeiss Axiocam digital camera (Carl Zeiss). For transmission electron microscopy (TEM) observations, clean diatom frustules were obtained boiling culture material for a few seconds with nitric (65%) and sulfuric (98%) acids (1:1:4, sample:HNO 3 :H 2 SO 4 ) to remove organic matter and washing it with distilled water (modified from Round et al., 1990). The material was then mounted on Formvar-coated grids and observed with a Philips 400 TEM (Philips Electron Optics BV). For scanning electron microscopy (SEM), material from successful mating experiments was fixed with glutaraldehyde (final concentration 2.5% v/v), placed on a filter in a Sweenex filter holder and dehydrated in a graded ethanol series (30%-100%). Filters were critical-point dried, mounted on stubs, sputter-coated with gold-palladium and observed with a JEOL JSM-6500F SEM (JEOL-USA Inc.).

| Toxin analysis
Cultures of P. allochrona strains SZN-B495 and SZN-B524 were grown in 1 L Erlenmeyer flasks (20°C, irradiance 70-80 μmol photon m −2 s −1 and 12:12 light:dark regime), harvested at their late exponential growth phase and centrifuged (750 g for 10 min). The cell pellet was stored at −18°C until analysis. Cells were lysed by sonication for 5 min, then added with 500 μl of MeOH/H 2 O (1:1) mix, and vortexed for 3 min. Cells were lysed by sonication again for 5 min and centrifuged at 1700 g for 5 min. The clear supernatant was transferred into a glass test tube, and the pellets were resuspended in 500 μl of MeOH/H 2 O (1:1) and vortexed for 3 min. All steps were repeated three times. Then, the supernatant was evaporated and the residue resuspended with 500 μl of MeOH/H 2 O (1:1). This solution was centrifuged at 7690 g for 5 min and finally 5 μl were analyzed by LC-MS/TOF. Certified standard of domoic acid (DA) was purchased from the National Research Council of Canada (NRCC).
Acetonitrile, methanol, and water were HPLC grade. Trifluoroacetic acid was obtained from VWR International (USA). The LC consisted of an Agilent 1100 instruments equipped with binary pump and an autosampler. Phenomenex Luna 3 μ PFP(2) (150 × 2.00 mm) was used for chromatographic separation. The isocratic mobile phase consisted of a mixture of 0.02% aqueous trifluoroacetic acid and acetonitrile in the ratio 90:10 (v/v), isocratic elution of 10% B at 0-15 min. The flow rate was 0.2 ml min −1 . Sample solutions (8, 4, 2, and 0.4 ppm) were prepared in ACN/W (1:9), and 5 μl was injected. The MS/TOF analysis worked in positive ion mode, and mass range was set at m/z 100-1000 u at a resolving power of 10,000.
All the acquisition and analysis of data were controlled by Agilent LC-MS TOF Software (Agilent). Tuning mix (G1969-85001) was used for lock mass calibration in our assay. Under these conditions, major peaks of DA would appear as the protonated ion at m/z 312, being accompanied by minor peaks consisting of sodium-binding ions at m/z 334. For the reference DA material, LOD is 0.001 μg kg −1 (1 ng) and LOQ 0.01 μg kg −1 (10 ng).

| Molecular analyses
Exponentially growing cultures were filtered on 0.8 μm pore size Isopore membrane filters (Millipore). Genomic DNA was extracted using the CTAB buffer as in (Tesson et al., 2011) and used as a template for the amplification of the following loci: partial 18S rDNA using Euk-A and Euk-B primers (Medlin et al., 1988); hypervariable (D1/D2) 28S rDNA region using DIR and D3Ca primers (Orsini et al., 2002); ITS rDNA using ITS-1 and ITS-4 primers (White et al., 1990); partial RUBISCO (rbcL) using rbcL1 and rbcL7 primers (Amato et al., 2007). Details of analyses carried out on individual strains are found in Table S1. PCRs were carried out in a PTC-200 Peltier Thermal Cycler (MJ Research) using reaction conditions as in the above-cited references for each of the amplified loci. The amplified fragments were purified using a QIAquick PCR purification kit (Qiagen Genomics) following the manufacturer's instructions, sequenced with the BigDye Terminator Cycle Sequencing technology (Applied Biosystems) and analyzed on an Automated Capillary Electrophoresis Sequencer "3730 DNA Analyzer" (Applied Biosystems).
Pseudo-nitzschia sequences retrieved from GenBank for each marker (Table S2) were aligned with sequences of P. allochrona using MAFFT (Katoh & Standley, 2013), with the L-INS-i option.
Cylindrotheca fusiformis and Cylindrotheca sp. were used as outgroup for 28S and rbcL, respectively, whereas Fragilariopsis curta and F. cylindrus were used as outgroups for 18S. Because the ITS region is highly variable, no outgroups were included in the analysis, in order to avoid ambiguous positions in the alignment. Maximum-likelihood (ML) was used for all markers. The substitution model used for each marker was selected through the Bayesian information criterion (BIC) and Akaike information criterion (AIC) implemented in MEGA X (Kumar et al., 2018). Details of the phylogenetic parameters used per each marker can be found in the legend of Figure S1. ML analyses were performed in MEGA X and trees were built with 1000 bootstrap replicates. All the phylogenetic trees were visualized using the interactive online tool iTOL (https://itol.embl. de, Letunic & Bork, 2019).
The analysis of the net evolutionary divergence, that is, the number of base substitutions per site between ITS sequences of P. allochrona and of the most closely related species, was conducted in MEGA XP. Standard error estimates were obtained through a bootstrapping procedure with 1000 replicates.
The ITS2 secondary structure was predicted for sequences from strains SZN-B509 (Gulf of Naples) and SZN-B495 (Ionian Sea) for P. allochrona and NerD1 for P. arenysensis using RNA structure (Reuter & Mathews, 2010) with suboptimal structure parameters set as follows: maximum% energy difference 10, maximum number of structures 20, and window size 5. Format conversion (CT format to dot-bracket format) was performed with RNApdbee (Antczak et al., 2014) and the 2D structures were drawn with VARNAv3.9 (Darty et al., 2009). The helices were labeled according to Mai and Coleman (1997). Compensatory base changes (CBCs) detection was performed with 4SALE v1.7 (Seibel et al., 2008), and hemicompensatory base changes (H-CBCs) and other polymorphisms were observed manually.

| Mating experiments
Sexual reproduction experiments were conducted on 18 exponentially growing cultures of P. allochrona isolated in July 2016 (Table S1). Prior to the experiments, the apical axis of 20 cells of each strain was measured in the light microscope (LM). One hundred and fifty-three couples of strains were mixed at concentrations of about 2000 cells per ml, each couple in a well of six-well culture plates containing 4 ml of F/2 medium, which were incubated at the conditions described above. The mixed cultures were examined daily using Zeiss Axiovert 200 light microscopes (Carl Zeiss). The content of some wells where sexual reproduction was taking place was fixed and prepared for SEM observations. Following a convention, the female mating type (−) was attributed to the strains that produced non-motile gametes, which were recognized as gametangia holding the zygote. By crossing strains with different cell sizes, it was possible to identify the zygote-bearing strain from its size and consider it as "female." This allowed to identify as "female mating type" all other strains sexually incompatible with that one and as male mating types (+) the remainder.
Sexual compatibility was tested by crossing two strains of opposite mating type (BB16 and CM63) of P. arenysensis between them and with each of four strains of P. allochrona (MC1028-B5, 8C3, MC1028-C5, and MC1029A2) as described above. Simultaneous data for environmental variables (temperature, salinity, and nutrients) were collected and quality controlled as described in Sabia et al. (2019).

| Ecological analysis
Based on the collection dates of more than 250 strains over 10 years and the recurring seasonal patterns observed (see results), P. delicatissima-like morphs occurring in summer and autumn were arbitrarily assigned to P. allochrona, while those recorded in winter and spring were assigned to other P. delicatissima-like species, which included P. delicatissima sensu stricto, P. arenysensis, and P. dolorosa.
Samples collected in late spring-early summer 2014, during an anomalous bloom not attributable to either P. allochrona or the other species based on recurrent seasonality, were excluded from the niche analysis. The environmental niches of P. allochrona and the other P.
delicatissima-like morphs were explored with the co-inertia analysis Outlying Mean Index (OMI; Doledec et al., 2000), which generates ordination axes that maximize the separation between species oc-  delicatissima-complex, that is, P. arenysensis and P. delicatissima.
The size of the 18 strains of P. allochrona tested in breeding experiments ranged between 22 and 54 μm (38.5 ± 4.2 μm; n = 364) in apical axis. All strains mated in numerous successful crosses that allowed to identify two groups of 10 and 8 strains of opposite mating types (Table S3) The net evolutionary divergence in ITS between P. allochrona and P. arenysensis (0.042 ± 0.010) was lower than that with P. micropora (0.082 ± 0.013) and P. delicatissima (0.090 ± 0.011; Table S4).
Toxin analyses of strains SZN-B524 and SZN-B495 did not reveal the presence of domoic acid.
At the LTER-MC station, P. delicatissima-like species usually showed a first bloom period from March through May and a second one from late June through mid-September (Figure 5a,b), with minima generally in late spring and late autumn-winter but F I G U R E 3 Maximum-likelihood phylogenies of the P. delicatissimacomplex species living in sympatry in the Gulf of Naples. Excerpts from Figure S1 representing the complete phylogenetic trees. (a) 18S; (b) 28S; (c) ITS; and (d) rbcL.
The new species P. allochrona is well separated in all markers from the closely related species that occur in the Gulf of Naples, namely, P. delicatissima, P. dolorosa, and P. arenysensis, and it is sister to P. arenysensis in all supported phylogenies, being closer to P. delicatissima only in the non-supported 18S phylogeny.  with the other P. delicatissima-like morphs. In either conditions, longer days appeared to favor more numerous and intense blooms ( Figure 5c,d). Over more than 30 years of sampling at the LTER-MC site, densities of P. delicatissima-like morphs occurring in summerautumn, here attributed to P. allochrona, were null (1984)(1985) or very low (1986)(1987)(1988)(1989)

| DISCUSS ION
Several results of this study support P. allochrona as a new cryptic species within the P. delicatissima-complex. Morphologically undistinguishable by definition from some closely related species, namely P. delicatissima and P. arenysensis, its distinctiveness is clearly seen in the molecular signature of three nuclear and one chloroplast sequences that are commonly used for species delimitation in diatoms.
Further, marked differences and conformational changes in the ITS secondary structure compared with its closest relative P. arenysensis and the failure of sexual reproduction experiments indicate mating incompatibility between the two sister species (Amato et al., 2007;Coleman, 2009). In Thalassiosira rotula, changes in the genetic structure among populations also correlate strongly with water temperature at the spatial and temporal scale (Whittaker & Rynearson, 2017), whereas a salinity gradient drives spatial patterns of genetic diversity in the case of Skeletonema marinoi in the Baltic Sea (Sjoqvist et al., 2015).
The relationship between neutral and functional genetic diversity within and among species is complex (Orsini et al., 2013), but the adaptive response under new environmental condition can be very rapid in diatoms (Pargana et al., 2020;Schaum et al., 2018). In the picoprasinophyte Ostreococcus, the tiniest eukaryote, cryptic sister species occupy different temporal and spatial niches (Limardo et al., 2017) and also show profound functional genomic differences (Palenik et al., 2007). In our case, biochemical differences between P. allochrona (as P. cf. delicatissima) and two of its closely related species, P. arenysensis and P. delicatissima, have been found in lipoxygenase enzymes mediating the metabolism of eicosapentaenoic acid (Lamari et al., 2013). Differences between P. arenysensis and P. delicatissima have also been described at the whole transcriptome level (Di Dato et al., 2015), which altogether provide a first indication of functional differences within this group of cryptic species.
Morphological identity and relatively small phylogenetic distance between P. arenysensis and P. allochrona, along with differences in their ecological and temporal niches, rouse some speculations on possible modes of speciation. Based on the lack of detection, followed by low abundance values, of summer-autumn P. delicatissimamorphs in the first years of the time series, P. allochrona could be a warm water species recently introduced in the Mediterranean Sea or in the Gulf of Naples, where it has found its optimal niche in summer.
At least two other cases of sudden appearance have been recorded in the Gulf of Naples time series, namely P. multistriata in 1995 and Skeletonema tropicum in 2002 (Zenetos et al., 2010). However, differently from the latter cases, the area where P. allochrona could have been introduced from cannot be traced. Despite numerous and detailed studies focusing on the genus Pseudo-nitzschia in the Mediterranean Sea and all over the world, so far P. allochrona has only been found in the Gulf of Naples (Lamari et al., 2013;McDonald et al., 2007;Ruggiero et al., 2015), Ionian Sea (this paper) and more recently in the Adriatic Sea (Arapov et al., 2020;Giulietti et al., 2021;Pugliese et al., 2017). In the latter area, P. allochrona (as P. cf. arenysensis) has been found in summer-autumn, like in the Gulf of Naples and Ionian Sea, while P. arenysensis has never been detected. The high spatial and taxonomic resolution offered by metabarcoding data could help clarifying the biogeography of these cryptic species, allowing to detect them in remote areas and thus supporting allopatric speciation, but the present distribution would hardly reflect the distribution at the time of speciation (Hendry, 2009). In addition, identical sequences may be shared by different taxa over the global scale for the usual marker for metabarcoding, the 18 s rDNA-V4 region (Piganeau et al., 2011), thus requiring that species presence recorded by these approaches be confirmed by isolation/cultivation studies or the use of more variable markers.
Hence, allopatric speciation can hardly be demonstrated for these cryptic microorganisms. Although it cannot be ruled out either, as an alternative mechanism it is tempting to postulate that the separation of the sister species P. allochrona and P. arenysensis may have occurred in sympatry in the Gulf of Naples. Habitat heterogeneity promotes species diversity at both the ecological and evolutionary time scale, preventing competitive exclusion and providing new niches to be occupied by different, co-existing species, eventually leading to ecological speciation (Chesson & Warner, 1981;Hutchinson, 1961). Whereas spatial partitioning of ecological conditions is hard to conceive in the planktonic realm, especially for microalgae confined to the photic zone, environmental factors can vary considerably along the year in strongly seasonal environments, such as the Mediterranean Sea, whereby time can replace space in creating the habitat heterogeneity required for ecological divergence, thus leading to allochronic speciation. A similar example of divergence by time in the same genus is offered by Pseudo-nitzschia galaxiae, a species that blooms in the Gulf of Naples in three different periods of the year with populations of three size classes (Cerino et al., 2005). The size classes actually correspond to distinct ribotypes (McDonald, 2007) that are also retrieved as distinct in eDNA metabarcoding studies . Whether the three P.
galaxiae populations are separate species needs further investigation, but the coherent pattern observed in size ranges, genetics and timing of the blooms provides a further possible case of isolation by time and allochronic speciation processes.
Genetic divergence among populations occurring at different times has been observed in various microalgal groups (Lebret et al., 2012;Richlen et al., 2012;Rynearson et al., 2006;Sassenhagen et al., 2018;Tammilehto et al., 2016;Whittaker & Rynearson, 2017), suggesting that temporal segregation could be a general mechanism for speciation in marine protists. Yet, phenology, that is, the seasonal time window of species' occurrences, has rarely been considered a stable, endogenous character in phytoplankton, whereby the alternation of different species over the seasons is postulated to be strictly driven by changes in environmental conditions.
Nevertheless, annually recurrent patterns are seen in long-term analysis of nano-and microphytoplankton species of the Gulf of Naples, where photoperiod is the most important explanatory variable driving community turnover (Longobardi et al., 2022;Ribera d'Alcalà et al., 2004). The mechanisms underlying allochronic speciation, and speciation in general, are not easy to clarify (Orsini et al., 2013), as both neutral and selective processes could be involved. Time and environmental variables covary, making it difficult to discern their respective contribution to the patterns observed in this study.
One possibility is that ecological segregation arises as a consequence of phenological shifts in the timing of the maximum abundance, a mechanism that would reduce competition for resources among individuals within a population (Devaux & Lande, 2008).
While most individuals respond rapidly and similarly to the relevant environmental cues, phenological characters often follow a skewed distribution (Forrest & Miller-Rushing, 2010), the long tail implying that a small part of the population may experience new environmental conditions during key life-cycle steps, such as reproduction. In such conditions that promote assortative mating, reduced gene flow can eventually lead to reproductive isolation between groups (Hendry & Day, 2005). This situation is somewhat analogous to a founder effect, where a subsample of the population colonizes new niches, diverging from the mother population via genetic drift (Barton & Charlesworth, 1984).
Phenological variations could be favored by the phenotypic plasticity that is typical of microalgae and/or by rapid adaptation to new ecological conditions (Schaum et al., 2018), or by selective processes acting on the standing intraspecific diversity (Orsini et al., 2013), which is also quite ample in phytoplankton populations (Rengefors et al., 2017).
Separation by time could be rapid enough as to be observable on decadal time scales, as no clear-cut boundary exists between the timescales of ecological and evolutionary processes (Carroll et al., 2007;Hendry et al., 2007). Contemporary evolution could be traced especially in microbial organisms, which are characterized by high growth rates and a few to several tens of generations over a single bloom season. In fact, swift genetic variations may take place in phytoplankton (Collins et al., 2014;Rengefors et al., 2017), where interannual variations in the genetic structure were actually observed in another species of the genus, P. multistriata (Tesson et al., 2014) which showed intermittent periods of weak and strong intraspecific diversity over the same bloom season (Ruggiero et al., 2018).
Not different from terrestrial plants, unicellular aquatic phototrophs can capture information from light and possess genes that are involved in the regulation of biological rhythms at various scales (Annunziata et al., 2019;Fortunato et al., 2015). Circa-annual rhythms and regular phenological patterns associated with photoperiodic response suggest that these microorganisms may also be able to measure the time of the year (Anderson & Keafer, 1987;Lambert et al., 2019). In this perspective, diversity in biological rhythms of plankton would be an optimal substrate for their evolutionary changes, contributing to isolation by time and speciation. In support

CO N FLI C T O F I NTE R E S T
We declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Molecular data produced in this study are available in GenBank.